Molecular Characterization of an SV Capture Site in the Mid-Region of the Presynaptic CaV2.1 Calcium Channel C-Terminal

Abstract

Neurotransmitter is released from presynaptic nerve terminals at fast-transmitting synapses by the action potential-gating of voltage dependent calcium channels (CaV), primarily of the CaV2.1 and CaV2.2 types. Entering Ca2+ diffuses to a nearby calcium sensor associated with a docked synaptic vesicle (SV) and initiates its fusion and discharge. Our previous findings that single CaVs can gate SV fusion argued for one or more tethers linking CaVs to docked SVs but the molecular nature of these tethers have not been established. We recently developed a cell-free, in vitro biochemical assay, termed SV pull-down (SV-PD), to test for SV binding proteins and used this to demonstrate that CaV2.2 or the distal third of its C-terminal can capture SVs. In subsequent reports we identified the binding site and characterized an SV binding motif. In this study, we set out to test if a similar SV-binding mechanism exists in the primary presynaptic channel type, CaV2.1. We cloned the chick variant of this channel and to our surprise found that it lacked the terminal third of the C-terminal, ruling out direct correlation with CaV2.2. We used SV-PD to identify an SV binding site in the distal half of the CaV2.1 C-terminal, a region that corresponds to the central third of the CaV2.2 C-terminal. Mutant fusion proteins combined with motif-blocking peptide strategies identified two domains that could account for SV binding; one in an alternatively spliced region (E44) and a second more distal site. Our findings provide a molecular basis for CaV2.1 SV binding that can account for recent evidence of C-terminal-dependent transmitter release modulation and that may contribute to SV tethering within the CaV2.1 single channel Ca2+ domain.

Early Metazoan Origin and Multiple Losses of a Novel Clade of RIM Presynaptic Calcium Channel Scaffolding Protein Homologs

“…On a more granular level, we observed apparent sequence divergence from a class I PDZ ligand among Ca V 1 channels in arthropods and Ca V 1 .1 channels in vertebrates, and a conserved hydrophobic insert disrupting the D/E-D/E/H-WC- COOH like motif in nematodes of the clade Rhabditomorpha ( Caenorhabditis , Haemonchus , and Strongyloides ), but not Trichinellida. Furthermore, we note the apparent absence of D/E-D/E/H-WC- COOH like motifs in the Ca V 2 channel of early-diverging chordate Ciona intestinalis and avian P/Q-type Ca V 2 .1 channels, the latter concomitant with the reported loss of C-terminal exon 47 (human equivalent) in the gene from Gallus gallus ( Snidal et al. 2018 ).…”

Conserved biophysical features of the CaV2 presynaptic Ca2+ channel homologue from the early-diverging animal Trichoplax adhaerens

“…Previously, we noted that the TCa V 2 channel lacks an acidic amino acid motif at its extreme C terminus with a consensus sequence of (D/E)(D/E/H)WC-COOH, which is conserved in cnidarian and bilaterian Ca V 2 channels and essential for interactions with the PDZ domains of presynaptic scaffolding proteins Mint ( 38 , 120 , 121 ) and RIM ( 49 , 122 , 123 ). TCa V 2 also lacks additional C-terminal motifs, upstream of the extreme C terminus, associated with Ca V 2 channel presynaptic localization and/or function ( 10 , 124 , 125 , 126 ). With respect to the Ca 2+ nanodomain arrangement, RIM has received considerable attention, because it has the capacity to directly interact with Ca V 2 channels and the vesicular protein Rab3 ( 127 ), and its genetic deletion in both vertebrates and invertebrates causes reduced localization of Ca V 2 channels at the synapse active zone and disrupted synaptic transmission ( 36 , 122 , 123 , 128 ).…”

“…However, this does not preclude the possibility that other redundant pre-synaptic interactions are present and conserved, where for example, RIM can interact with the calcium channel CaVβ subunit (129), and another CaV2 channel-binding protein, RIM-BP (36). Furthermore, additional interactions that might operate independently of RIM (124)(125)(126) could also be conserved, should the apparently absent CaV2 channel protein interaction motifs be divergent, as observed for ligands of protein interaction domains such as SH3 (130).…”

Early metazoan origin and multiple losses of a novel clade of RIM pre-synaptic calcium channel scaffolding protein homologues

“…On a more granular level, we observed apparent sequence divergence from a class I PDZ ligand among Ca V 1 channels in arthropods and Ca V 1.1 channels in vertebrates, and a conserved hydrophobic insert disrupting the D/E-D/E/H-WC- COOH like motif in nematodes of the clade Rhabditomorpha ( Caenorhabditis, Haemonchus and Stronglyloides ), but not Trichinellida. Furthermore, we note the apparent absence of D/E-D/E/H-WC- COOH like motifs in the Ca V 2 channel of early-diverging chordate Ciona intestinalis and avian P/Q-type Ca V 2.1 channels, the latter concomitant with the reported loss of C-terminal exon 47 (human equivalent) in the gene from Gallus gallus (Snidal, et al 2018). To corroborate our sequence data, we also predicted C-terminal PDZ ligands with the cluster based prediction tool PDZPepInt (Kundu, et al 2014), which references a set of 226 PDZ domains from humans, mouse, fly and nematode worm, finding that most metazoan and all pre-metazoan Ca V s bear predicted PDZ ligands (Figure 5A).…”